The field of the invention is related to compositions and methods useful for preventing graft rejection in a recipient following organ transplantation.
Organ transplantation has been used to improve the quality of human life. Substantial progress has been made in the transplantation of kidneys, hearts, lung, livers and pancreas. Current immunosuppressive drugs are generally effective in blocking the immediate rejection of these organs. However, when the organ is from an unrelated donor, i.e., allograft, these drugs become less successful with the passage of time because immunosuppressive drugs are often ineffective in blocking chronic allograft rejection. In addition, there are significant side effects associated with long term immunosuppressive therapy. Each year approximately 10,000 kidney transplants are performed in the United States. While the chances that the graft will function well for at least one year have been increasing, there has been a lack of progress in preventing chronic allograft rejection during the past 20 years (See FIG. 1; In Fundamental Immunology, 4th ed., Paul, W. E. (ed.), Lippincott-Raven, Philadelphia, 1999, p. 1201). As a result, only 50% of transplants are still functioning years later. There is an urgent need, therefore, for new methods to prevent chronic rejection.
Graft rejection occurs when the immune system of the recipient recognizes foreign histocompatibility antigens. Infrequently, rejection is caused by antibodies, either preformed or the result of multiple blood transfusions. Rejection generally occurs when T lymphocytes from the recipient recognize and respond to donor histocompatibility antigens (Pescovitz M D, Thistlethwaite J R Jr, Auchincloss H Jr, et al. J Exp Med 1984;160:1495-1508).
There are two major histocompatibility complex (MHC) loci. Both major and minor histocompatibility antigens have been described as well as the genes that encode them. One encodes MHC class I antigens which are recognized by CD8+ T cells and another encodes MHG class II antigens which are recognized by CD4+ cells. MHC class I antigens are expressed on almost all tissues of the body. Both MHC I and II antigens are very polymorphic so that it is highly unlikely that antigens from unrelated individuals will be identical.
Differences in MHC antigens between donor and recipient trigger a strong immune response by the recipient which results in the rejection of the transplanted organ. Foreign MHC antigens are directly recognized by the recipient""s immune cells and also indirectly recognized by antigen-presenting cells of the recipient which have processed donor MHC antigens. The classical model of allograft rejection emphasizes CD4+ T cells of the recipient recognizing MHC class II antigens of the donor. These activated CD4+ cells serve as helper cells for recipient CD8+ which are sensitized by direct recognition of donor MHC class I antigens. The activated CD8+ cells then kill donor cells by lysing them (Mizuochi T, Golding H, Rosenberg A S, Glimcher L H, Malek T R, Singer A. J Exp Med 1985;162:427-443. 205). Further studies have revealed additional participation of recipient antigen presenting cells, B cells, NK cells and NK T cells which adds complexity to the mechanisms responsible for graft rejection.
Graft destruction which occurs within the first few weeks after transplantation is called xe2x80x9cacute rejectionxe2x80x9d. Usually, the use of immunosuppressive drugs temporarily prevents this result. Unfortunately, the grafts may eventually fail weeks or months later. This failure is referred to as xe2x80x9cchronic rejection.xe2x80x9d Both humoral and cellular mechanisms have been implicated in chronic rejection. Anti-donor antibodies have been claimed to promote chronic rejection, but this is controversial. It is generally believed that chronic rejection is the consequence of persistent sensitization of the immune system to donor MHC antigens. The immune cells of the recipients cannot xe2x80x9clearnxe2x80x9d to accept the donor MHC antigens as self and respond by attacking the graft.
There are two approaches to prevent graft rejection. The first is by treatment with non-specific immunosuppressants and the second is to induce donor-specific tolerance. The standard first approach is to use immunosuppressive drugs such as steroids, azathioprine, mycophenolate, cyclosporine, FK-506, rapamycin, leflunomide, or 15-deoxyspergualin. These drugs suppress immune responses by inhibiting lymphocyte gene transcription, cytokine signal transduction, nucleotide synthesis and cell differentiation. These drugs are associated with lifelong increased risks of infection and malignancy. In addition, anti-T cell antibodies such as anti-lymphocyte serum or anti-thymocyte globulin are also powerful immunosuppressants. However, they have major side effects include serum sickness and infectious complications More recently, OKT3, a mouse antibody directed against the CD3 antigen of humans, has become widely used in clinical transplantation. (Cosimi A B, Burton R C, Colvin R B, et al Transplantation 1981,32:535-539). Other monoclonal antibodies used include the antibody to the IL-2 receptor (anti-CD25) and the anti-ICAM-1 or anti-TNF-xcex1 to block the effector mechanism of graft rejection. These monoclonal antibodies also have broad toxic side effects.
The ultimate goal of transplantation immunology is to enable the recipient to become tolerant to donor histocompatibility antigens. That is, to prevent the recipient""s immune cells from recognizing donor antigens (i.e., accepting the donor organ as xe2x80x9cselfxe2x80x9d) so that the graft is not rejected. The current state of the art in this area is reviewed herein and elsewhere (See Hugh Auchincloss, Jr., Megan Sykes, and David H. Sachs In Fundamental Immunology, 4th ed., Paul, W. E. (ed.), Lippincot-Raven, Philadelphia, N.Y., 1999 pp 1182-1222).
Tolerance can be achieved by three mechanisms. The first is xe2x80x9cclonal deletionxe2x80x9d; the elimination of lymphocytes which react to the donor antigens. The second is xe2x80x9cclonal anergyxe2x80x9d; the failure of T cells to proliferate in response to donor antigen. Anergy is generally reversible and can be reversed by infection or elimination of antigen (Rocha B, Tanchot C, Von Boehmer H. J Exp Med 1993;177:1517-1521) (Ramsdell F, Fowlkes B J. Science 1992;257:1130-1134). The third is xe2x80x9csuppressionxe2x80x9d; which can be either non-specific or antigen-specific. Non-specific suppression can result from the secretion of soluble molecules that inhibit immune function. Suppressive molecules include prostaglandins (Snijdewint F G M, Kalinski P, Wierenga E A, Bos J D, Kapsenberg M I. J Immunol 1993;150:5321-5329, Betz M, Fox B S. J Immunol 1991;146:108-113), nitric oxide (Langrehr J M, Dull K E, Ochoa J B, et al. Transplantation 1992;53:632-640), and cytokines (Verbanac K M, Carver F M, Haisch C E, Thomas J M. Transplantation 1994;57:893-900); Raju G P, Belland S E, Eisen H J. Transplantation 1994;58:392-396).
Certain T cells, called xe2x80x9csuppressor cellsxe2x80x9d, produce inhibitory cytokines which include IL-4, IL-10, and TGF-xcex2 which, non-specifically, block graft rejection (Qin L, Chavin K D, Ding Y, Woodward J E, Favaro J P, Lin J, Bromberg J S. Ann Surg 1994;220:508-519); (Qin L, Chavin K D, Ding Y, et al. J Immunol 1996; 156:2316-2323); (Zheng X X, Steele A W, Nickerson P W, Steurer W, Steiger J, Strom T B. J Immunol 1995;154:5590-5600). The existence of alloantigen-suppressor cells have been reported (Pearce N W, Spinelli A, Gurley K E, Hall B M. Transplantation 1993;55:374-379; Roser B J. Immunol Rev 1989; 107:179-202; Tomita Y, Mayumi H, Eto M, Nomoto K. J Immunol 1990;144: 463-473), but these cells are difficult to clone (Koide J, Engleman E G. J Immunol 1990;144:32-40).
Naturally occurring suppressor T cells produced by the thymus have been characterized in mice. These are CD4+ cells that express CD25, cell surface IL-2 receptor a chains (Shevach, E. A. (2000) Annu. Rev. Immunol. 18:423-449; Seddon, B., and D. Mason, (2000) 21.95-99; Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, and M. Toda, (1995) J. Immunol. 155:1151-1164). To date, this T cell subset has not been well described in humans and whether these cells can be expanded in the periphery is unknown.
CD4+ cells repeatedly stimulated with IL-10 or activated with immature dendritic cells develop down-regulatory activity (Groux, H., A. O""Garra, M. Bigler, M. Rouleau, S. Antojejko, J. E. De Vries, and M. G. Roncarolo, (1997) Nature. 389:737-742; Jonuleit, H., E. Schmitt, G. Schuler, K. Jurgen, and A. H. Enk. (2000) J Exp Med 192:1213-1222). These T cells, called TR1 or TR1-like cells, are anergic and their immunosuppressive effects are mediated by IL-10 and TGF-xcex2. Anergic T cells suppress other T cell responses by targeting antigen-presenting cells (Taams, L. S., A. J. M. L. van Rensen, M. C. M. Poelen, C. A. C. M. van Els, A. C. Besseling, J. P. A. Wagenaar, W. van Eden, and M. H. M. Wauben (1998) Eur J Immunol 28:2902-2912; Vendetti, S., J. G. Chai, J. Dyson, E. Simpson, G. Lombardi and R. Lechler, (2000), J. Immunol. 165:1175-1181). Unfortunately, large numbers of these cells are required for suppressive activity and their capacity to expand is very poor.
The profile of cytokines produced by T cells can affect the survival of an organ graft. The shift in T cell response from the pro-inflammatory Th1 response (IL-2 and IFN-xcex3) to the anti-inflammatory Th2 (IL-4, IL-10) response has been associated with allograft acceptance (Roser B J. Immunol Rev 989: 107:179-202; Lancaster F, Chui Y L, Batchelor J R. Nature 1985;315:336-337; Wilson. Immunol Rev 1989;107:159-176). Only limited data, however, implicate an active role of Th2 cells in tolerance induction (Bucy R P, Li J, Huang G Q, Honjo K, Xu X Y. [Abstract]. FASEB J 1995;9:A497; Wilson. Immunol Rev 1989;107:159-176). Moreover, in some cases Th2 cells can mediate or contribute to graft rejection.
Methods to specifically direct T cells to become tolerant or produce inhibitory cytokines would be very helpful in promoting the survival of transplanted organs. Several tolerance-inducing strategies have been attempted in combination with conventional immunosuppressive drugs. In rodents, tolerance can be achieved by giving a dose of lethal irradiation to a mouse, and saving the animal by giving back T cell-depleted syngeneic and allogeneic bone marrow cells. The hematopoietic cells that repopulate the animal will display histocompatibility antigens of both donor and recipient cells and, therefore, will be tolerant to grafts from each mouse strain (Singer A, Hathcock K S, Hodes R J. J Exp Med 1981;153:1286). Non-myeloablative conditioning regimens have been described where mice are sublethally irradiated, T cell depleted with monoclonal antibodies and given either anti-CD154 or CTLA4lg which block co-stimulatory molecules (Wekerle, 1999). This strategy achieves central tolerance and should have long lasting effects, but has not yet been performed in large animals. None of these strategies have been used to replace chronic therapy in clinical transplantation.
Peripheral tolerance can be achieved by blocking co-stimulatory molecules. The combination of CTLA4lg and anti-CD154 markedly prolongs the survival of primary skin allografts in mice (Larsen C P, Elwood E T, Alexander D Z, et al. Nature 1996;381:434-438; Kirk A D, Harlan D M, Armstrong N N, et al. Proc Natl Acad Sci USA 1997;94:8789-8794). A major problem with strategies to block co-stimulatory molecules is that they cannot prevent generation of new T cells in the recipient capable of recognizing donor antigens.
There are examples of solid organ transplants that have survived for many years in human recipients who did not receive hematopoletic cell transplants (Starzl T E, et al. (1993) Transplantation, 55:1272-1277). In these instances, passenger leukocytes from the graft might emigrate to the thymus and tolerize subsequently developing thymocytes. The heavy doses of immunosuppressive drugs used to prevent acute rejection blocks this thymic education. Strategies to reduce the dosage of immunosuppressive therapy might overcome this problem and lead to long lasting central tolerance.
An ideal strategy to prevent graft rejection would be to induce T cells to develop the capacity to suppress the immune attack by the recipient against donor histocompatibility antigens. Although CD4+ cells repeatedly activated in the presence of IL-10 develop potent suppressor activity, these cells have a very short life span and poor proliferative potential (Groux H, et al., (1997) Nature, 389:737-42). Thus, there is a need for a method to generate suppressor T cells which are hardy and able to proliferate.
In accordance with the objects outlined above, the present invention provides compositions and methods that can be used to induce T cell tolerance in a solid organ transplant recipient. The compositions include compounds that inhibit or suppress immune function by inducing a population of T cells to develop suppressor activity. Compounds useful in the compositions of the invention include anti-inflammatory cytokines, such as IL4, IL-10 and TGF-xcex2, chemokines, prostaglandins and nitric oxide.
In an additional aspect, the present invention provides methods of inhibiting graft rejection in a recipient comprising removing peripheral blood mononuclear cells (PBMC) from a donor and recipient, culturing the donor and recipient cells together in the presence of a compound that induces T cell suppressor activity, and administering these treated cells to the recipient following graft transplantation
In a further aspect, the invention uses closed systems for the purification, conditioning and expansion of T cell populations before administering them to a patient.